EP0846764A2 - Famille de facteurs alpha neurotrophiques des cellules gliales - Google Patents

Famille de facteurs alpha neurotrophiques des cellules gliales Download PDF

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Publication number
EP0846764A2
EP0846764A2 EP97309375A EP97309375A EP0846764A2 EP 0846764 A2 EP0846764 A2 EP 0846764A2 EP 97309375 A EP97309375 A EP 97309375A EP 97309375 A EP97309375 A EP 97309375A EP 0846764 A2 EP0846764 A2 EP 0846764A2
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Prior art keywords
polypeptide
seq
nucleotide sequence
receptor
amino acid
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EP97309375A
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EP0846764A3 (fr
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Geoffrey M.P. SmithKline Beecham Pharm. Lawrence
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SmithKline Beecham Ltd
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SmithKline Beecham Ltd
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Priority claimed from GBGB9624677.2A external-priority patent/GB9624677D0/en
Priority claimed from GBGB9709463.5A external-priority patent/GB9709463D0/en
Application filed by SmithKline Beecham Ltd filed Critical SmithKline Beecham Ltd
Publication of EP0846764A2 publication Critical patent/EP0846764A2/fr
Publication of EP0846764A3 publication Critical patent/EP0846764A3/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to newly identified polynucleotides, polypeptides encoded by them and to the use of such polynucleotides and polypeptides, and to their production. More particularly, the polynucleotides and polypeptides of the present invention relate to GDNF alpha receptor family, hereinafter referred to as GDNF alpha 3 receptor. The invention also relates to inhibiting or activating the action of such polynucleotides and polypeptides.
  • the receptor complex for GDNF has been identified by a number of groups and shown to comprise the orphan tyrosine kinase receptor Ret (M.Trupp et al., Nature 381, 785-789, 1996; P. Durbec et al., Nature 381, 789-793, 1996) and an ancilliary component, GDNF-alpha (J. Treanor et al., Nature 382, 80-83, 1996 and S. Jing et al., Cell 85 1113-1124, 1996).
  • GDNF alpha receptor modulates GDNF stimulated signalling of the Ret receptor, which may be essential since Ret is a proto-oncogene. More recently, a GDNF related polypeptide called Neurturin has been identified (P. Kotzbauer et al., Nature 384, 467-470, 1996). It's receptor components however remain unknown. This raises the possibility of further alpha receptors being identified and possibly additional Ret-like receptors. In addition other GDNF-like homologues may yet be identified, thereby forming a family of trophic polypeptides and receptor complexes.
  • the invention relates to GDNF alpha 3 receptor polypeptides and recombinant materials and methods for their production.
  • Another aspect of the invention relates to methods for using such GDNF alpha 3 receptor polypeptides and polynucleotides.
  • Such uses include the treatment of neurodegenerative diseases (such as Parkinson's Disease, amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), Huntington's Disease, Alzheimer's Disease, diabetic neuropathy), muscular diseases (including the muscular dystrophies) and nerve and muscle trauma, among others.
  • the invention relates to methods to identify agonists and antagonists using the materials provided by the invention, and treating conditions associated with GDNF alpha 3 receptor imbalance with the identified compounds.
  • Yet another aspect of the invention relates to diagnostic assays for detecting diseases associated with inappropriate GDNF alpha 3 receptor activity or levels.
  • the present invention relates to GDNF alpha 3 receptor polypeptides.
  • the GDNF alpha 3 receptor polypeptides include the polypeptides of SEQ ID NO:2; as well as polypeptides comprising the amino acid sequence of SEQ ID NO:2; and polypeptides comprising the amino acid sequence which have at least 80% identity to that of SEQ ID NO:2 over the entire length of SEQ ID NO:2, and still more preferably at least 90% identity, and even still more preferably at least 95% identity to SEQ ID NO: 2. Furthermore, those with at least 97-99% are highly preferred.
  • GDNF alpha 3 receptor polypeptides having the amino acid sequence which have at least 80% identity to the polypeptide having the amino acid sequence of SEQ ID NO: 2 over its entire length, and still more preferably at least 90% identity, and even still more preferably at least 95% identity to SEQ ID NO: 2. Furthermore, those with at least 97-99% are highly preferred.
  • GDNF alpha 3 receptor polypeptides exhibit at least one biological activity of the receptor.
  • Fragments of the GDNF alpha 3 receptor polypeptides are also included in the invention.
  • a fragment is a polypeptide having an amino acid sequence that entirely is the same as part, but not all, of the amino acid sequence of the aforementioned GDNF alpha 3 receptor polypeptides.
  • fragments may be "free-standing," or comprised within a larger polypeptide of which they form a part or region, most preferably as a single continuous region.
  • Representative examples of polypeptide fragments of the invention include, for example, fragments from about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, and 101 to the end of GDNF alpha 3 receptor polypeptide.
  • “about” includes the particularly recited ranges larger or smaller by several, 5, 4, 3, 2 or 1 amino acid at either extreme or at both extremes.
  • fragments characterized by structural or functional attributes such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions.
  • Other preferred fragments are biologically active fragments.
  • Biologically active fragments are those that mediate receptor activity, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Also included are those that are antigenic or immunogenic in an animal, especially in a human.
  • GDNF alpha 3 receptor polynucleotides further include a polynucleotide comprising a nucleotide sequence that has at least 80% identity to a nucleotide sequence encoding the GDNF alpha 3 receptor polypeptide of SEQ ID NO:2 over the entire coding region, and a polynucleotide that is at least 80% identical to that having SEQ ID NO: 1 over its entire length.
  • polynucleotides at least 90% identical are particularly preferred, and those with at least 95% are especially preferred.
  • those with at least 97% are highly preferred and those with at least 98-99% are most highly preferred, with at least 99% being the most preferred.
  • GDNF alpha 3 receptor polynucleotides are a nucleotide sequence which has sufficient identity to a nucleotide sequence contained in SEQ ID NO: 1 to hybridize under conditions useable for amplification or for use as a probe or marker.
  • the invention also provides polynucleotides which are complementary to such GDNF alpha 3 receptor polynucleotides.
  • GDNF alpha 3 receptor of the invention is structurally related to other proteins of the GDNF alpha receptor family, as shown by the results of sequencing the cDNA encoding human GDNF alpha 3 receptor.
  • the cDNA sequence of SEQ ID NO:1 contains an open reading frame (nucleotide number 1 to 1200) encoding a polypeptide of 400 amino acids of SEQ ID NO:2.
  • the amino acid sequence of SEQ ID NO:2 has about 36% identity (using Bestfit) in 400 amino acid residues with GDNF alpha receptor (J. Treanor et al., Nature, 382, 80-83, 1996; S. Jing et al., Cell, 85, 1113-1124, 1996).
  • One polynucleotide of the present invention encoding GDNF alpha 3 receptor may be obtained using standard cloning and screening, from a cDNA library derived from mRNA in cells of human testes, fetal heart and skeletal muscle using the expressed sequence tag (EST) analysis (Adams, M.D., et al. Science (1991) 252:1651-1656; Adams, M.D. et al., Nature, (1992) 355:632-634; Adams, M.D., et al., Nature (1995) 377 Supp:3-174).
  • Polynucleotides of the invention can also be obtained from natural sources such as genomic DNA libraries or can be synthesized using well known and commercially available techniques.
  • nucleotide sequence encoding GDNF alpha 3 receptor polypeptide of SEQ ID NO:2 may be identical to the polypeptide encoding sequence hereinbefore described (nucleotide number 1 to 1200 of SEQ ID NO:1), or it may be a different sequence, which as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID NO:2.
  • the polynucleotide may include the coding sequence for the mature polypeptide or a fragment thereof, by itself; the coding sequence for the mature polypeptide or fragment in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence, or other fusion peptide portions.
  • a marker sequence which facilitates purification of the fused polypeptide can be encoded.
  • the present invention further relates to polynucleotides that hybridize to the herein above-described sequences.
  • the present invention especially relates to polynucleotides which hybridize under stringent conditions to the herein above-described polynucleotides.
  • stringent conditions means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences.
  • Polynucleotides of the invention which are identical or sufficiently identical to a nucleotide sequence contained in SEQ ID NO:1 or a fragment thereof, may be used as hybridization probes for cDNA and genomic DNA, to isolate full-length cDNAs and genomic clones encoding GDNF alpha 3 receptor and to isolate cDNA and genomic clones of other genes that have a high sequence similarity to the GDNF alpha 3 receptor gene.
  • hybridization techniques are known to those of skill in the art.
  • these nucleotide sequences are 80% identical, preferably 90% identical, more preferably 95% identical to that of the referent.
  • the probes generally will comprise at least 15 nucleotides.
  • such probes will have at least 30 nucleotides and may have at least 50 nucleotides. Particularly preferred probes will range between 30 and 50 nucleotides.
  • polynucleotides and polypeptides of the present invention may be employed as research reagents and materials for discovery of treatments and diagnostics to animal and human disease.
  • host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present invention.
  • Introduction of polynucleotides into host cells can be effected by methods described in many standard laboratory manuals, such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY (1986) and Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL , 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) such as calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection.
  • bacterial cells such as streptococci, staphylococci, E. Coli, Streptomyces and Bacillus subtilis cells
  • fungal cells such as yeast cells and Aspergillus cells
  • insect cells such as Drosophila S2 and Spodoptera Sf9 cells
  • animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells
  • plant cells include bacterial cells, such as streptococci, staphylococci, E. Coli, Streptomyces and Bacillus subtilis cells
  • fungal cells such as yeast cells and Aspergillus cells
  • insect cells such as Drosophila S2 and Spodoptera Sf9 cells
  • animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells
  • Such systems include, among others, chromosomal, episomal and virus-derived systems, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.
  • viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses
  • vectors derived from combinations thereof such as those derived from plasmid and bacteriophage genetic elements, such as cosmid
  • the expression systems may contain control regions that regulate as well as engender expression.
  • any system or vector suitable to maintain, propagate or express polynucleotides to produce a polypeptide in a host may be used.
  • the appropriate nucleotide sequence may be inserted into an expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL ( supra ).
  • secretion signals may be incorporated into the desired polypeptide. These signals may be endogenous to the polypeptide or they may be heterologous signals.
  • the polypeptide be produced at the surface of the cell.
  • the cells may be harvested prior to use in the screening assay. If GDNF alpha 3 receptor polypeptide is secreted into the medium, the medium can be recovered in order to recover and purify the polypeptide; if produced intracellularly, the cells must first be lysed before the polypeptide is recovered.
  • GDNF alpha 3 receptor polypeptides can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification. Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification.
  • This invention also relates to the use of GDNF alpha 3 receptor polynucleotides for use as diagnostic reagents. Detection of a mutated form of GDNF alpha 3 receptor gene associated with a dysfunction will provide a diagnostic tool that can add to or define a diagnosis of a disease or susceptibility to a disease which results from under-expression, over-expression or altered expression of GDNF alpha 3 receptor. Individuals carrying mutations in the GDNF alpha 3 receptor gene may be detected at the DNA level by a variety of techniques.
  • Nucleic acids for diagnosis may be obtained from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy material.
  • the genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR or other amplification techniques prior to analysis.
  • RNA or cDNA may also be used in similar fashion. Deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to labeled GDNF alpha 3 receptor nucleotide sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures.
  • DNA sequence differences may also be detected by alterations in electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing. See, e.g., Myers et al., Science (1985) 230:1242. Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and S1 protection or the chemical cleavage method. See Cotton et al., Proc Natl Acad Sci USA (1985) 85: 4397-4401.
  • an array of oligonucleotides probes comprising GDNF alpha 3 receptor nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of e.g., genetic mutations.
  • the diagnostic assays offer a process for diagnosing or determining a susceptibility to neurodegenerative diseases (such as Parkinson's Disease, amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), Huntington's Disease, Alzheimer's Disease, diabetic neuropathy) and muscular diseases (including the muscular dystrophies) through detection of mutation in the GDNF alpha 3 receptor gene by the methods described.
  • neurodegenerative diseases such as Parkinson's Disease, amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), Huntington's Disease, Alzheimer's Disease, diabetic neuropathy
  • muscular diseases including the muscular dystrophies
  • Assay techniques that can be used to determine levels of a protein, such as an GDNF alpha 3 receptor, in a sample derived from a host are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays.
  • the nucleotide sequences of the present invention are also valuable for chromosome identification.
  • the sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome.
  • the mapping of relevant sequences to chromosomes according to the present invention is an important first step in correlating those sequences with gene associated disease. Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library).
  • genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes).
  • linkage analysis coinheritance of physically adjacent genes.
  • the differences in the cDNA or genomic sequence between affected and unaffected individuals can also be determined. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease.
  • the novel GDNF alpha 3 receptor has been mapped to chromosome 5q31 by radiation hybrid mapping.
  • Several disease loci map to this position including Limb-girdle muscular dystrophy type 1A, autosomal nonsyndromic sensoneural deafness and corneal dystrophy, granular type.
  • polypeptides of the invention or their fragments or analogs thereof, or cells expressing them can also be used as immunogens to produce antibodies immunospecific for the GDNF alpha 3 receptor polypeptides.
  • immunospecific means that the antibodies have substantiall greater affinity for the polypeptides of the invention than their affinity for other related polypeptides in the prior art.
  • the above-described antibodies may be employed to isolate or to identify clones expressing the polypeptide or to purify the polypeptides by affinity chromatography.
  • Antibodies against GDNF alpha 3 receptor polypeptides may also be employed to treat neurodegenerative diseases (such as Parkinson's Disease, amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), Huntington's Disease, Alzheimer's Disease, diabetic neuropathy), muscular diseases (including the muscular dystrophies) and nerve and muscle trauma, among others.
  • neurodegenerative diseases such as Parkinson's Disease, amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), Huntington's Disease, Alzheimer's Disease, diabetic neuropathy
  • muscular diseases including the muscular dystrophies
  • nerve and muscle trauma among others.
  • Yet another aspect of the invention relates to a method of inducing immunological response in a mammal which comprises, delivering GDNF alpha 3 receptor polypeptide via a vector directing expression of GDNF alpha 3 receptor polynucleotide in vivo in order to induce such an immunological response to produce antibody to protect said animal from diseases.
  • composition which, when introduced into a mammalian host, induces an immunological response in that mammal to a GDNF alpha 3 receptor polypeptide wherein the composition comprises a GDNF alpha 3 receptor polypeptide or GDNF alpha 3 receptor gene.
  • the vaccine formulation may further comprise a suitable carrier. Since GDNF alpha 3 receptor polypeptide may be broken down in the stomach, it is preferably administered parenterally (including subcutaneous, intramuscular, intravenous, intradermal etc. injection).
  • Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation instonic with the blood of the recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use.
  • the vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.
  • the GDNF alpha 3 receptor polypeptide of the present invention may be employed in a screening process for compounds which bind the receptor and which activate (agonists) or inhibit activation of (antagonists) the receptor polypeptide of the present invention.
  • polypeptides of the invention may also be used to assess the binding of small molecule substrates and ligands in, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures. These substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics. See Coligan et al., Current Protocols in Immunology 1(2):Chapter 5 (1991).
  • GDNF alpha 3 receptor polypeptides are responsible for many biological functions, including many pathologies. Accordingly, it is desirous to find compounds and drugs which stimulate GDNF alpha 3 receptor on the one hand and which can inhibit the function of GDNF alpha 3 receptor on the other hand.
  • agonists are employed for therapeutic and prophylactic purposes for such conditions as neurodegenerative diseases (such as Parkinson's Disease, amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), Huntington's Disease, Alzheimer's Disease, diabetic neuropathy), muscular diseases (including the muscular dystrophies) and nerve and muscle trauma.
  • Antagonists may be employed for a variety of therapeutic and prophylactic purposes for such conditions as neurodegenerative diseases (such as Parkinson's Disease, amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), Huntington's Disease, Alzheimer's Disease, diabetic neuropathy), muscular diseases (including the muscular dystrophies) and nerve and muscle trauma.
  • neurodegenerative diseases such as Parkinson's Disease, amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), Huntington's Disease, Alzheimer's Disease, diabetic neuropathy
  • muscular diseases including the muscular dystrophies
  • such screening procedures involve producing appropriate cells which express the receptor polypeptide of the present invention on the surface thereof.
  • Such cells include cells from mammals, yeast, Drosophila or E. coli.
  • Cells expressing the receptor (or cell membrane containing the expressed receptor) are then contacted with a test compound to observe binding, or stimulation or inhibition of a functional response.
  • Examples of potential GDNF alpha 3 receptor antagonists include antibodies or, in some cases, oligonucleotides or proteins which are closely related to the ligand of the GDNF alpha 3 receptor, e.g., a fragment of the ligand, or small molecules which bind to the receptor but do not elicit a response, so that the activity of the receptor is prevented.
  • GDNF alpha 3 receptor polypeptides are administered to a subject an inhibitor compound (antagonist) as hereinabove described along with a pharmaceutically acceptable carrier in an amount effective to inhibit activation by blocking binding of ligands to the GDNF alpha 3 receptor, or by inhibiting a second signal, and thereby alleviating the abnormal condition.
  • soluble forms of GDNF alpha 3 receptor polypeptides still capable of binding the ligand in competition with endogenous GDNF alpha 3 receptor may be administered. Typical embodiments of such competitors comprise fragments of the GDNF alpha 3 receptor polypeptide.
  • expression of the gene encoding endogenous GDNF alpha 3 receptor can be inhibited using expression blocking techniques.
  • Known such techniques involve the use of antisense sequences, either internally generated or separately administered. See, for example, O'Connor, J Neurochem (1991) 56:560 in Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression , CRC Press, Boca Raton, FL (1988).
  • oligonucleotides which form triple helices with the gene can be supplied. See, for example, Lee et al., Nucleic Acids Res (1979) 3:173; Cooney et al., Science (1988) 241:456; Dervan et al., Science (1991) 251:1360. These oligomers can be administered per se or the relevant oligomers can be expressed in vivo.
  • the retroviral expression construct may then be isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention such that the packaging cell now produces infectious viral particles containing the gene of interest.
  • These producer cells may be administered to a subject for engineering cells in vivo and expression of the polypeptide in vivo.
  • Another approach is to administer a therapeutic amount of GDNF alpha 3 receptor polypeptides in combination with a suitable pharmaceutical carrier.
  • Polypeptides used in treatment can also be generated endogenously in the subject, in treatment modalities often referred to as "gene therapy" as described above.
  • cells from a subject may be engineered with a polynucleotide, such as a DNA or RNA, to encode a polypeptide ex vivo, and for example, by the use of a retroviral plasmid vector. The cells are then introduced into the subject.
  • a polynucleotide such as a DNA or RNA
  • Antibodies as used herein includes polyclonal and monoclonal antibodies, chimeric, single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other immunoglobulin expression library.
  • Isolated means altered “by the hand of man” from the natural state. If an "isolated” composition or substance occurs in nature, it has been changed or removed from its original environment, or both.
  • a polynucleotide or a polypeptide naturally present in a living animal is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated”, as the term is employed herein.
  • Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods.
  • Variant is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties.
  • a typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below.
  • a typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical.
  • a variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination.
  • a substituted or inserted amino acid residue may or may not be one encoded by the genetic code.
  • a variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis.
  • Identity is a measure of the identity of nucleotide sequences or amino acid sequences. In general, the sequences are aligned so that the highest order match is obtained. “Identity” per se has an art-recognized meaning and can be calculated using published techniques.

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EP97309375A 1996-11-27 1997-11-20 Famille de facteurs alpha neurotrophiques des cellules gliales Withdrawn EP0846764A3 (fr)

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GB9624677 1996-11-27
GBGB9624677.2A GB9624677D0 (en) 1996-11-27 1996-11-27 Novel compounds
GBGB9709463.5A GB9709463D0 (en) 1997-05-09 1997-05-09 Novel compounds
GB9709463 1997-05-09

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998054213A2 (fr) * 1997-05-30 1998-12-03 Amgen Inc. Recepteurs du facteur neurotrope
WO1999049039A2 (fr) * 1998-03-23 1999-09-30 Genentech, Inc. LE GFRα3 ET SES UTILISATIONS
WO1999058711A1 (fr) * 1998-05-13 1999-11-18 Garvan Institute Of Medical Research Procede servant a diagnostiquer ou a evaluer une predisposition a la maladie d'alzheimer
US6677135B1 (en) 1996-05-08 2004-01-13 Biogen, Inc. Ret ligand (RetL) for stimulating neutral and renal growth
US7026138B1 (en) 1998-03-23 2006-04-11 Genentech, Inc. Polynucleotides encoding GFRα3
US10058590B2 (en) 2000-09-14 2018-08-28 Toshikazu Nakamura Methods of treating amyotrophic lateral sclerosis by HGF

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Cited By (10)

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Publication number Priority date Publication date Assignee Title
US6677135B1 (en) 1996-05-08 2004-01-13 Biogen, Inc. Ret ligand (RetL) for stimulating neutral and renal growth
US6861509B1 (en) 1996-05-08 2005-03-01 Biogen, Inc. Antibodies to Ret and RetL3
WO1998054213A2 (fr) * 1997-05-30 1998-12-03 Amgen Inc. Recepteurs du facteur neurotrope
WO1998054213A3 (fr) * 1997-05-30 1999-01-07 Amgen Inc Recepteurs du facteur neurotrope
WO1999049039A2 (fr) * 1998-03-23 1999-09-30 Genentech, Inc. LE GFRα3 ET SES UTILISATIONS
WO1999049039A3 (fr) * 1998-03-23 2000-02-10 Genentech Inc LE GFRα3 ET SES UTILISATIONS
US7026138B1 (en) 1998-03-23 2006-04-11 Genentech, Inc. Polynucleotides encoding GFRα3
US7691973B2 (en) 1998-03-23 2010-04-06 Genentech, Inc. GFRα3 polypeptides
WO1999058711A1 (fr) * 1998-05-13 1999-11-18 Garvan Institute Of Medical Research Procede servant a diagnostiquer ou a evaluer une predisposition a la maladie d'alzheimer
US10058590B2 (en) 2000-09-14 2018-08-28 Toshikazu Nakamura Methods of treating amyotrophic lateral sclerosis by HGF

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CA2216625A1 (fr) 1998-05-27
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